I keep reading that cDNA obtained with random hexamers yield short cDNA fragments, which are fine for qPCR, while for longer targets, oligoDT should be used.
However, shouldn't these short fragments prime one another during the PCR, so that we get the (virtually) full cDNA representation of a transcriptome? I imagine that random hexamers would keep priming to the newly-generated DNA giving shorter fragments throughout the PCR, but does it impact the final product so much that random haxamers are not good for cDNA synthesis for cloning?
Specifically, I want to amplify a 1 kb product from cDNA to be inserted into a plasmid. Should it be feasible with random-primed cDNA, or would you recommend using oligoDT?
Thanks in advance.
Sonia, random hexamers and oligoDT are good to generation population libraries of things but are never going to get you a specific individual clone. The PCR process will also bias towards shorter fragments. Do you have the sequence for the 1kb product you want?
Hello and thank you. Michael.
And sorry, that was a mental shortcut indeed. I want to amplify a sequence from cDNA - and I do have specific primers for the gene. However, this is a difficult, GC-rich sequence and I have difficulties with this PCR, wich is done on a cDNA matrix. I know that the conditions for such PCR can be optimised, but I was wondering if the method of cDNA sythesis can also affect the efficacy of "the second" PCR, made on the cDNA. From what I have read, oligo DT would be preferable in this case, but I can't see its advantage over random hexamers.
One advantage of oligo DT is that you have one fixed end and it is highly enriched for mRNA sequences due to the requirement that one end will be the poly A, neither of these will be true for random hexamers. However if the problem you are having is that the PCR reaction with your defined primers is not working, possibly due to high GC, then using random primers would likely have the same high GC amplification problem and your desired sequence would be underrepresented in that population of products.
I would either work to optimize your PCR reaction, or if you have the sequence of the segment you want why not just have it synthesized. The cost is probably much less than a few weeks of your time trying to get everything to work.
Thank you! That makes much sense. Also, the expression of the gene I want to amplify is not especially high, and by being GC-rich, its fraction in total cDNA, not only from mRNAs, must be significantly lower. I'll consider other options than a PCR, then. Thanks a lot, that was really helpful.
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